Amonafide salts

ABSTRACT

Disclosed is a salt of amonafide or amonafide analogs represented Structural Formula (I):  
                 
 
     R1 is —(CH 2 ) n N + HR3R4 X −  or R1 is —(CH 2 ) n N + HR3R4 X −  or —(CH 2 ) n NR3R4 when R2 is —N + HR6R7.  
     R2 is —OR5, halogen, —NR6R7, —N + HR6R7 X −,  sulphonic acid, nitro, —NR5COOR5, —NR5COR5 or —OCOR5;  
     R3 and R4 are independently H, C1-C4 alkyl group or, taken together with the nitrogen atom to which they are bonded, a non-aromatic nitrogen-containing heterocyclic group;  
     each R5 is independently —H or a C1-C4 alkyl group;  
     R6 and R7 are independently H, C1-C4 alkyl group or, taken together with the nitrogen atom to which they are bonded, a non-aromatic nitrogen-containing heterocyclic group;  
     n is an integer from 0-3; and  
     X −  is the carboxylate anion of an organic carboxylic acid compound.  
     Also disclosed are methods of preparing certain compounds represented by Structural Formula (I).

RELATED APPLICATION

[0001] This application is a continuation-in-part of InternationalApplication No. PCT/US03/12619, which designated the United States andwas filed Apr. 22, 2003, published in English, which is acontinuation-in-part of and claims priority to U.S. Ser. No. 10/128,129,filed Apr. 22, 2002. The entire teachings of the above applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Traditional pharmaceutical process technology for manipulatingthe physical properties and water solubility of these importantanti-cancer drug moieties has been to render them water soluble withstrong mineral acids. Salt formation is reserved as the final step inthe synthesis, as described by Brana and associates (U.S. Pat. No.5,420,137; 1995) more as an after thought for formulation purposes thanas an integral or even strategic component, of the reaction synthesis.Such mono and divalent mineral acid salts retain hygroscopicity, and thedivalent species, which form hydrates, have also been found to beincompatible with many pharmaceutical auxiliaries required for preparingsterile injectables, tablets or gelatin capsules.

[0003] In contradistinction to the prior art and accepted practice, wehave found that the early incorporation of organic acids into thesynthetic elaboration of amonafide and its aminoalkyl analogs moietiespermits more rapid isolation and purification of intermediates, higherconcentrations of reactants during the synthetic process, and moredesirable properties, such as bulk density, flocculence andcompressibility.

[0004] Furthermore, the resulting organic salts show higher solubilitiesin water as well as in osmotically balanced electrolyte solutions, whichotherwise would be incompatible via common ion effects with inorganic,mineral acid salts such as the hydrochlorides and methylsulfonates usedroutinely heretofore. Moreover, organic acid salts of the presentinvention retain a higher degree of amphiphilic compatibility both withprotic and aprotic solvents of varying polarity, thereby affording abroader range of crystallizing conditions for purposes of purificationand isolation than would be afforded by the corresponding mineral acidsalts.

[0005] Examination of the process chemistry for amonafide readilyillustrates the shortcomings of the prior art and the adverse propertiesof the resulting mineral salts, which have been circumvented by thepresent invention. Among the synthetic approaches described in thepatent and professional literature, the common denominator requiresacylation of 1-amino-2,2-N,N-dimethylamino ethylene diamine, or itssimilarly substituted homologs, with a polycyclic, substituted arylanhydride as shown in FIG. 1. Thus for amonafide, in accordance with themethod of Brana and Sanz (Eur. J. Med. Chem 16:207, 198.1) compounds Iand II in FIG. 1 are combined in ethanol to afford a precipitate ofmitonafide, which must then be recrystallized multiple times from alarger volume of ethanol to be freed of tarry-black or brownby-products.

[0006] While the acylation may be conducted at a concentration of 1 gramof precursor anhydride in 25 ml of solvent, recrystallization ofmitonafide requires three recrystallizations at a concentration of 1 grin 75 ml to afford light cream colored material, free of tarrysubstances and exhibiting a constant melting. Although the initialyields according this process range within 60-80 percent, subsequentpurification reduces the net yield to 30% of material with sufficientpurity for subsequent conversion into a pharmaceutically acceptableend-product.

[0007] These isolation and purification conditions also apply to thesynthesis of mitonafide analogs in toluene followed by precipitationwith excess gaseous hydrochloric acid, as described by Zee-Cheng andCheng (U.S. Pat. No. 4,665,071; 1987). The mitonafide hydrochloride, ofunspecified stoichiometry and hydration, obtained by this reaction is areddish brown precipitate, containing 12% by weight of tar with nodifferential solubility between water and alcohol, again requiringmultiple recrystallizations to afford a hydrochloride salt material ofsuitable quality for pharmaceutical use.

[0008] In the present invention, as shown in FIG. 1, it has now beenfound that the isolation of the mitonafide moiety (III) from theethanolic reaction mixture is facilitated by admixture and completedissolution of a suitable organic carboxylic acid compound, which uponcooling affords a near colorless adduct upon crystallization from amother liquor which retains the preponderance of colored impuritieswhich might otherwise have co-crystallized as in the case of the priorart. In contrast to the synthetic approach of Brana and associates (Eur.J. Med. Chem 16:207, 1981; U.S. Pat. No. 4,204,063; 1980), describedhereinabove, the mitonafide is obtained directly as an organic salt, inthe first step of the synthesis for the target amonafide compound,rather than post facto in accordance with the alternate teachings as inU.S. Pat. No. 5,420,137 (1995) relating to the monohydrochloride ormonomethylsulfate salts of amonafide obtained by controlled titration ofamonafide itself at the end of the reaction sequence.

[0009] Those skilled in the art will also recognize that salt formationof mitonafide as an isolation step as taught by U.S. Pat. No. 4,665,071(1987) cannot be construed as an obvious precedent for the instantinvention insofar as organic acids, which are the salt forming reagentsin this invention are known to be insoluble in toluene, and similarnon-polar solvents, even at reflux. Unlike gaseous HCl used inaccordance with the prior art on mitonafide salt isolation, the organicacids of this invention are solids which can be metered with accuracy soas to achieve a precise titration stoichiometry, an elusive objectivewhen the dispensing of gaseous acids is called for as the prevailingalternative.

[0010] The novelty of the invention described here, when contrasted toprior art, is further affirmed by the unexpected finding concerning thecatalytic hydrogenolysis of the nitro substituents in the mitonafidestructural skeleton.

[0011] In the specific prior art on the formation of amonafide salts,congeneric with Stucture IV in FIG. 1, Brana and associates (U.S. Pat.No. 5,420,137; 1995) fail to describe the properties of the precursormitonafide nor do they describe the method of hydrogenation to theresulting amonafide free base. However, in prior disclosures (SpanishPatent 533,542; 1983) on a method for the industrial productionspecifically of amonafide, these same authors indicate that nitroreduction of the precursor mitonafide free base is effected with 10%palladium-on-carbon (Pd/C) via transfer hydrogenation in the presence ofexcess hydrazine under refluxing ethanolic conditions. This procedure isalso summarized as the preferred approach in the chemical reviewliterature by the same authors (Ars Pharmaceutica 36:377-415, 1995).

[0012] Those skilled in the art would recognize that such an approachcould not be practiced if the mitonafide precursor were composed of apre-formed acid salt. Under such circumstances, one might reasonablyexpect that the hydrazine donor reagent would be neutralized by ionexchange and become unavailable as a substrate for diimide formation,which is the active reducing species catalyzed by Pd/C. In effect, anyfollowers of the teachings of Brana and associates would havecontemplated only the use of free-base precursors rather than resortingto the less obvious alternative, namely direct reduction of a mitonafidesalt as taught in this patent.

[0013] Within the larger scope of organic functional grouptransformations, those skilled in the art will recognize that thecatalytic hydrogenation of aryl nitro compounds to the correspondingsubstituted anilines is usually practiced in ethanol, mixtures ofethanol and water, or in the so called universal solvents (e.g.dimethylormamide and dimethylacetamide) which are resistant tohydrogenation. Respected, classical monographs on the subject by P. N.Rylander (Catalytic Hydrogenation in Organic Synthesis, New York:Academic Press, 1979) and M. Freifelder (Practical CatalyticHydrogenation, New York: Wiley, 1971) acknowledge that the solubility ofaryl nitro compounds in general precludes use of water as thehydrogenation medium.

[0014] These experts also indicate that the preferred source of protonsto effect suppression of the imine and oxime by-products of incompletehydrogenation is achieved by admixture of the substrate withhydrochloric acid. Even in the presence of mineral acids, hydrogenationsin water are poorly documented and considered idiosyncratic in both thetraditional and current hydrogenation laboratory and industrialpractice. Use of organic acids, such as acetic acid or formic acid, hasbeen described, but with the caveat that dehydrative acylation willoccur, thus affording the corresponding N-acyl aryl-amines asyield-lowering contaminants.

[0015] Thus, in the context of this invention, neither the specificliterature on amonafide synthesis nor on methods of hydrogenation can becited as precedent for the non-obvious chemical manipulations which werefound to be advantageous here. First, the use of organic carboxylic acidcompounds to effect purification and isolation of mitonafide and itsanalogs has not been described heretofore. Second, application oforganic carboxylic acid salts of mitonafide and its analogs as directprecursors for catalytic hydrogenation has not be considered orpromulgated as an effective practice. Third, the high degree of watersolubility of these organic salts, itself an unexpected phenomenon,coupled with the reluctance among experts to recommend catalytichydrogenations in water as the sole solvent, would have precludedexploration of the novel approach presented here.

[0016] Beyond these issues which demonstrate non-obviousness, thereexist further practical advantages to the use of organic carboxylic acidcompounds, and especially their preferred analogs, the organiccarboxylic diacid compounds, in the context of this invention. Theresulting aralkyl naphthalimide salts show water solubilites as high as1:1 by proportional admixture in contrast to the mono or divalent saltsof hydrochloric, methanesulfonic, or of other mineral acids, whosesolubilities fall below 10% by weight. Bulk processing is facilitatedfor purposes of industrial synthesis, filtration, purification, anddispensing of dosage units prior to sterile filtration andlyophilization.

[0017] In dry form, these organic carboxylic acid salts show higher bulkdensity, porosity and compaction than their analogs mineral acid salts,while presenting lower hygroscopicity. Thus, they are more suitable forprocessing by direct pressing, rather than solely by granulation oragglomeration.

[0018] In terms of biological burden, the organic carboxylate anionspresent no electrolyte load, unlike the mineral acid anions, and arebiodegradable through normal cellular pathways of intermediarymetabolism. In either the case of inorganic or organic acid anions, itis a matter of record that these species provide charge balance,solubility, mechanical, adsorptive or absorptive properties to the drugmoiety to which they are attached. However, it is known throughout thepractice of medicinal and pharmaceutical chemistry that the salt formper se will not affect the pharmacological activity, which in the caseof the polycyclic aryl and aralkylamine containing intercalator drugs istheir antitumor action.

[0019] For example, in reviewing the current pharamacopoeia, theintercalator drug ametantrone is known to be equally active as the freebase, hydrochloride, monoacetate and diacetate, the difference in thesalt form being their bulk formulation properties. Its analog,NSC-639366, on the other hand is being developed preclinically as thefumarate salt, in preference to the hydrochloride or acetate. In thecase of asulacrine, the preferred salt form is the isethionate. Forcrisnatol and exatecan, it is the mesylate which is pharmaceuticallymost suitable. Many other drug classes, with diverse modes of action,have been developed as salts of organic acids. For example, the L-malatesalts of clebopride, an antinausea medication, of almotriptan, anantimigraine medication, and of pizotifen, an antihistamine, haveexhibited enhanced solubility without alteration of their respectivemedicinal properties.

SUMMARY OF THE INVENTION

[0020] One embodiment of the present invention is a compound representedby Structural Formula (I):

[0021] R1 is —(CH₂)_(n)N⁺HR3R4 X⁻ or R1 is —(CH₂)_(n)N⁺HR3R4 X⁻ or—(CH₂)_(n)NR3R4 when R2 is —N⁺HR6R7.

[0022] R2 is —OR5, halogen, —NR6R7, —N⁺HR6R7 sulphonic acid, nitro,—NR5COOR5, —NR5COR5 or —OCOR5.,

[0023] R3 and R4 are independently H, C1-C4 alkyl group or, takentogether with the nitrogen atom to which they are bonded, a non-aromaticnitrogen-containing heterocyclic group.

[0024] Each R5 is independently —H or a C1-C4 alkyl group.

[0025] R6 and R7 are independently H, a C1-C4 alkyl group or, takentogether with the nitrogen atom to which they are bonded, a non-aromaticnitrogen-containing heterocyclic group.

[0026] n is an integer from 0-3.

[0027] X⁻ is the carboxylate anion of an organic carboxylic acidcompound. Examples of suitable organic carboxylic acids are providedbelow.

[0028] Another embodiment of the present invention is a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand a compound oh the present invention.

[0029] Another embodiment of the present invention is a method oftreating a subject with cancer. The method comprises administering tothe subject an effective amount of a compound of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 is a schematic showing the synthesis of amonafide malatesalt by the method disclosed herein. Many other organic carboxylic acidsalts of the amonafide structural skeleton can be prepared by the methoddisclosed herein.

[0031]FIG. 2 is a graph showing the percent net cell growth of cancercell from the cell lines H460 (♦), SF268 (▪) and MCF7 (▴) in thepresence of varying concentrations of amonafide malate. Theconcentration of amonafide malate is given in μM.

[0032]FIG. 3 is a graph showing the percent net cell growth of breastcancer cell from the cell lines MCF-7 (♦), BT474 (

), MDA-231 (▪), T47D (□) and SKBr3 (▴) using Sulforhodamine B analysisin the presence of varying concentrations of amonafide malate. Theconcentration of amonafide malate is given in μM.

[0033]FIG. 4 is a graph showing the percent net cell growth ofcolorectal cancer cell from the cell lines HT29(♦), HCT116 (▪) andCOLO205 (▴) using Sulforhodamine B analysis in the presence of varyingconcentrations of amonafide malate. The concentration of amonafidemalate is given in μM.

[0034]FIG. 5 is a graph showing the percent net cell growth of lungcancer cell from the cell lines H460 (▴) and H23 (▪) usingSulforhodamine B analysis in the presence of varying concentrations ofamonafide malate. The concentration of amonafide malate is given in μM.

[0035]FIG. 6 is a graph showing the percent net cell growth of lungcancer cell from the cell lines H460 (▴), H23 (▪) and A549 (▴) using MTTanalysis in the presence of varying concentrations of amonafide malate.The concentration of amonafide malate is given in μM.

[0036]FIG. 7 is a graph showing the percent net cell growth of prostatecancer cell from the cell lines DU-145 (♦), PC-3 (▪) and LNCaP (▴) usingSulforhodamine B analysis in the presence of varying concentrations ofamonafide malate. The concentration of amonafide malate is given in ±1M.

[0037]FIG. 8 is a graph showing the percent growth inhibition byamonafide malate of MCF-7, COLO205 and PC3 tumors in mice. Amonafidemalate was administered intraperitoneally (IP) or subcutaneously (SC).

[0038]FIG. 9 is a graph showing in vivo percent growth inhibition ofintraperitoneally implanted solid tumor by amonafide malate in mice.Cell lines used were MCF-7, MDA-231, H23 and COLO205. Amonafide malatewas administered intraperitoneally twice-daily at either 15 mg/kg or 29mg/kg.

[0039]FIG. 10 is a graph showing in vivo percent growth inhibition ofintraperitoneally implanted solid tumor by amonafide malate in mice.Cell lines used were MCF-7, MDA-231, H23 and COLO205. Amonafide malatewas administered intraperitoneally twice-daily at either 15 mg/kg or 29mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention is directed to organic carboxylic acidsalts of amonafide and organic carboxylic acid salts of amonafidederivatives and precursors represented by Structural Formula (I).Preferably in Structural Formula (1), n is 2; R3 and R4 are the same andare —H, —CH₃ or —CH₂CH₃; and R2 is —NO₂, —NH₂ or —NH₃ ⁺X⁻. Morepreferably, n is 2; R3 and R4 are —CH₃; and R2 is —NO₂, —NH₂ or —NH₃⁺X⁻. Suitable values for X⁻ are provided below.

[0041] The present invention is also directed to methods of preparingorganic acid salts of amonafide and derivatives represented byStructural Formula (II) by hydrogenating compounds represented byStructural Formula (III) in water. Preferably in Structural Formula (II)and (III), n is 2; R3 and R4 are the same and are —H, —CH₃ or —CH₂CH₃.More preferably, n is 2; and R3 and R4 are —CH₃. Suitable values for X⁻are provided below.

[0042] Most preferably, the present invention is directed to organiccarboxylic acid salts of amonafide and methods of preparation therefor.The structure of amonafide is represented by Structural Formula (IV):

[0043] The compounds disclosed herein with two amine groups, includingamonafide salts, can be monovalent, meaning that one of the amine groupsis protonated, or divalent, meaning that both amine groups areprotonated. A divalent compound can be protonated by two differentmonocarboxylic acid compounds (i.e., the two Xs in Structural Formula(I) represent two different monocarboxylic acid compounds), by two molarequivalents of the same monocarboxylic acid compound (i.e., the two Xsin Structural Formula (I) each represent one molar equivalent of thesame monocarboxylic acid compound), or by one molar equivalent of adicarboxylic acid compound (i.e., the two Xs in Structural Formula (I)together represent one dicarboxylic acid compound). Alternatively, threemolar equivalents a divalent compound are protonated by two molarequivalents of a tricarboxylic acid compound. All of these possibilitiesare meant to be included within Structural Formulas (I) and (IV) above.

[0044] An organic carboxylic acid compound is an organic compound havingone or more carbon atoms and a carboxylic acid functional group.Suitable organic carboxylic acid compounds for use in preparing thecompounds of the present invention are water soluble (typically a watersolubility greater than 20% weight to volume), produce water solublesalts with aryl amines and alkyl amines and have a pKa>2.0. Included arearyl carboxylic acids, aliphatic carboxylic acids (typically C1-C4),aliphatic dicarboxylic acids (typically C2-C6), aliphatic tricarboxylicacids (typically C3-C8) and heteroalkyl carboxylic acids. An aliphaticcarboxylic acid can be completely saturated (an alkyl carboxylic acid)or can have one or more units of unsaturation. A heteroalkyl carboxylicacid compound is an aliphatic carboxylic acid compound in which one ormore methylene or methane groups are replaced by a heteroatom such as O,S, or NH. Examples of heteroalkyl carboxylic acid compounds include aC1-C5 heteroalkyl monocarboxylic acid compound (i.e., a C2-C6 alkylmonocarboxylic acid compound in which one methylene or methane group hasbeen replaced with O, S or NH) and C3-C8 a heteroalkyl dicarboxylic acidcompound (i.e., a C2-C7 alkyl dicarboxylic acid compound in which onemethylene or methane group has been replaced with O, S or NH).

[0045] An aliphatic carboxylic acid compound can be straight orbranched. An aliphatic carboxylic acid can be substituted(functionalized) with, one or more functional groups. Examples include ahydroxyl group (e.g., a hydroxy C2-C6 aliphatic monocarboxylic acids, ahydroxy C3-C8 aliphatic dicarboxylic acid and a hydroxy C4-C10 hydroxyaliphatic tricarboxylic acid), an amine (e.g., an amino C2-C6 aliphaticmonocarboxylic acid, an amino C3-C8 aliphatic dicarboxylic acid and anamino C4-C10 aliphatic tricarboxylic acid), a ketone (e.g., a keto C2-C6aliphatic monocarboxylic acid, a keto C3-C8 dicarboxylic acid or a ketoC4-C10 tricarboxylic acid) or other suitable functional group.

[0046] Examples of suitable organic acids are:

[0047] saturated aliphatic monocarboxylic acids such as formic acid,acetic acid or propionic acid;

[0048] unsaturated aliphatic monocarboxylic acids such as 2-pentenoicacid, 3-pentenoic acid, 3-methyl-2-butenoic acid or 4-methyl-3-pentenoicacid;

[0049] functionalized acids such as hydroxycarboxylic acids (e.g. lacticacid, glycolic, pyruvic acid, mandelic acid);

[0050] ketocarboxylic acids (e.g. oxaloacefic acid andalpha-ketoglutaric acid);

[0051] amino carboxylic acids (e.g. aspartic acid and glutamic acid);

[0052] saturated aliphatic dicarboxylic acids such as malonic acid,succinic acid or adipic acid;

[0053] unsaturated aliphatic dicarboxylic acids such as maleic acid orfumaric acid;

[0054] functionalized di- and tricarboxylic acids such as malic acid,tartaric acid, citric acid gluconic acid.

[0055] aryl carboxylic acids having sufficient water solubility, e.g.,e.g., 4-hydroxybenzoic acid, salicylic acid, anthranilic acid, anisicacid and vanillic acid.

[0056] Non-aromatic nitrogen-containing heterocyclic rings arenon-aromatic nitrogen-containing rings which include zero, one or moreadditional heteroatoms such as nitrogen, oxygen or sulfur in the ring.The ring can be five, six, seven or eight-membered. Examples includemorpholinyl, thiomorpholinyl, pyrrolidinyl, piperazinyl, piperidinyl,azetidinyl, azacycloheptyl, or N-phenylpiperazinyl.

[0057] Hydrogenation of compounds represented by Structural Formula(III) is carried out under a hydrogen atmosphere at pressures between 5and 50 pounds per square inch (psi), preferably between 13 and 17 psi. Ahydrogenation catalyst is required, for example Pd/C, Pt/C, PtO₂, RaneyNickel and activated elemental iron or zinc. After hydrogenation, amonovalent compound is obtained. The corresponding divalent compound canbe obtained by reacting the product with an additional equivalent of thesame or different carboxylic acid compound.

[0058] The starting compound represented by Structural Formula (III) canbe prepared by reacting the corresponding free base, i.e., where R1 is—(CH₂)_(n)NR3R4, with an organic carboxylic acid compound. Preferably,the resulting product is crystallized before hydrogenating. The freebase can be prepared by reacting H₂N(CH₂)_(n)NR3R4 with3-nitro-1,8-nitronaphthalic anhydride, wherein n, R3 and R4 are asdefined in Structural Formula (I). Specific conditions for carrying outthis reaction are described in U.S. Pat. No. 4,204,063, the entireteachings of which are incorporated herein by reference.

[0059] The compounds disclosed herein are useful for the treatment of ina subject. A “subject” is a mammal, preferably a human, but can also bean animal in need of veterinary treatment, e.g., companion animals(e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs,horses, and the like) and laboratory animals (e.g., rats, mice, guineapigs, and the like). The compounds of the present invention can be usedto treat a broad spectrum of cancers, including carcinomas, sarcomas andleukemias. Examples of carcinomas, including adenocarcinomas that can betreated using the compounds' of the present invention are breast, colon,lung, kidney and prostate cancers. An example of sarcomas that can betreated using the compounds of the present invention are gliomas.Examples of leukemias that can be treated using the method include AcuteMyelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), AcuteLymphocytic Leukemia (ALL) and Chronic Lymphocytic Leukemia (CLL).Preferably, the cancers that are treated using the compounds of thepresent invention are breast, colorectal, lung and prostate cancers.

[0060] An “effective amount” is the quantity of compound in which abeneficial clinical outcome is achieved when the compound isadministered to a subject with a multi-drug resistant cancer. A“beneficial clinical outcome” includes a reduction in tumor mass, areduction in the rate of tumor growth, a reduction in metastasis, areduction in the severity of the symptoms associated with the cancerand/or an increase in the longevity of the subject compared with theabsence of the treatment. The precise amount of compound administered toa subject will depend on the type and severity of the disease orcondition and on the characteristics of the subject, such as generalhealth, age, sex, body weight and tolerance to drugs. It will alsodepend on the degree, severity and type of cancer. The skilled artisanwill be able to determine appropriate dosages depending on these andother factors. Effective amounts of the disclosed compounds fortherapeutic application typically range between about 0.35 millimolesper square meter of body surface area (mmole/msq) per day and about 2.25millimoles per square meter of body surface area (mmole/msq) per day,and preferably between 1 mmole/msq and 1.5 mmole/msq on five day cyclesby intravenous infusion.

[0061] The disclosed compounds are administered by any suitable route,including, for example, orally in capsules, suspensions or tablets or byparenteral administration. Parenteral administration can include, forexample, systemic administration, such as by intramuscular, intravenous,subcutaneous, or intraperitoneal injection. The compounds can also beadministered orally (e.g., dietary), topically, by inhalation (e.g.,intrabronchial, intranasal, oral inhalation or intranasal drops), orrectally, depending on the type of cancer to be treated. Oral orparenteral administration are preferred modes of administration.

[0062] The disclosed compounds can be administered to the subject inconjunction with an acceptable pharmaceutical carrier as part of apharmaceutical composition for treatment of cancer. Formulation of thecompound to be administered will vary according to the route ofadministration selected (e.g., solution, emulsion, capsule). Suitablepharmaceutical carriers may contain inert ingredients which do notinteract with the compound. Standard pharmaceutical formulationtechniques can be employed, such as those described in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Suitablepharmaceutical carriers for parenteral administration include, forexample, sterile water, physiological saline, bacteriostatic saline(saline containing about 0.9% mg/ml benzyl alcohol), phosphate-bufferedsaline, Hank's solution, Ringer's-lactate and the like. Methods forencapsulating compositions (such as in a coating of hard gelatin orcyclodextrasn) are known in the art (Baker, et al., “Controlled Releaseof Biological Active Agents”, John Wiley and Sons, 1986).

EXEMPLIFICATION

[0063] The method of the invention is illustrated in more detail by thefollowing examples which are not intended to be limiting in any way. Thecourse of the organic acid salt formation of amonafide and analogsthereof can be followed via the determination of the products formed bymeans of chromatography and NMR spectroscopy. The formed salts arefurther characterized by mass spectrometry and by elementary analysis.

Example 1 Direct Synthesis of Intercalator Drug Amonafide As An OrganicAcid (L-Malate) Salt by the Method of the Present Invention

[0064] The following synthetic scheme is meant to describe in detail thereaction shown in FIG. 1.

[0065] 1. Preparation of Mitonafide Malate (III, FW 447.42)

[0066] Reactants:

[0067] (I) 3-nitro-1,8-nitronaphthalic anhydride, (FW 243.18, CAS3027-38-1, purity 99%, ACROS, cat. # 27873-0250);

[0068] (II) N,N-dimethylethylenediamine (FW 88.15, CAS 108-00-9, purity99%, ACROS cat. # 11620-100);

[0069] L-malic acid (FW 134.09, CAS 97-67-6, purity 99% ACROS cat. #15059-1000

[0070] Synthetic Procedure:

[0071] 100 gr. (0.41 mol, 1 eq.) of the anhydride (I) were combined with1300 ml of anhydrous ethanol in a 3L 3-neck round bottom flask fittedwith an adding funnel and mechanical paddle stirrer. While vigorouslystirring the suspension, a solution of 40 gr (0.45 mol, 1.1 eq.) of thediamine (II) in 100 ml of anhydrous ethanol was added as a rapid drip.Stirring was continued for 12 hours (overnight), and thereafter themixture was brought to reflux for 1 hour.

[0072] Upon cooling to an internal temperature of 80° C., a pre-warmed(also to 80° C.) solution of 60 gr. (0.42 mol, 1.09 eq.) of L-malic acidin 100 ml of ethanol was added in one portion to the reaction flask andstirring continued for 3 hours. Stirring was stopped and when thereaction reaches room temperature, the crude mitonafide malate wasrecovered by filtration. The solids were resuspended in 1 liter ofanhydrous isopropanol, refiltered, and rinsed with diethyl ether. Theywere then transferred to a drying dish, triturated mechanically andvacuum desiccated in a drying oven at 0.1 Torr with heating to 30° C.for 12 hours.

[0073] A tan solid (160 gr., 87% yield) was obtained, mp 160-162° C.,with a proton NMR spectrum (in perdeuterated acetic acid, 80 MHz)conforming to theory: malate-CH₂, 2.8 ppm, asymmetric doublet, 2H;N,N-(CH₃)₂, 3.1 ppm, singlet, 6H; imido-N—CH₂, 3.7 ppm, degeneratetriplet, 2H; amino-N—CH₂ and malate-CH, 4.6 ppm, multiplet, 3H; aryl-CH,8-9.3 ppm, two apparent triplets and doublet, 5H; OH, 11.4 ppm, singlet,3H.

[0074] Material prepared in this manner was sufficiently pure for use insubsequent synthetic steps.

[0075] Alternatively, for analytical and biological samples,recrystallization was achieved by suspending the product in aqueousethanol (water/ethanol 1/5 v/v per gram of III), heating to boil andremoving any insolubles by hot filtration. Upon cooling, the mass wasfiltered, rinsed with diethyl ether and vacuum desiccated to affordlight tan, birefringent plates (140 gr, 76% overall yield), mp 163-164°C., homogenous by HPLC, under the conditions shown in Table 1.

[0076] 2. Preparation of Amonafide Malate (IV FW 417.42

[0077] Synthetic Procedure:

[0078] A solution of 134 gr. (0.3 mole) mitonafide malate (III) wassuspended in 1 liter of deionized, degassed water under an argon blanketin a Parr hydrogenation pressure bottle. 1.4 gr. of 10% Pd/C are added,and the mixture was then evacuated and purged with hydrogen gas (threetimes), then connected to a Parr apparatus and pressurized with hydrogengas to 15 psi. The reaction was vigorously shaken at room temperaturebecoming a yellow solution instead of a tan colored suspension withintwo hours. It was left to hydrogenate for an additional 12 hours(overnight).

[0079] After evacuative removal of the hydrogen headspace andreplacement with nitrogen, the reaction mixture was stirred with 40 gr.of activated carbon, warmed to 50° C., and passed in a Buchner funnelthrough filter paper overlayed with pre-washed Celite filtration aid.The filtrate was concentrated in a 2 liter round bottom flask, underreduced pressure in a rotary evaporator with a heating bath thermostatedto 50° C. When a thin crust had begun to form along the meniscus of thesyrupy concentrate, which weighed between 240-250 gr, the flask wasrefrigerated (4° C.) for 12 hrs (overnight) to permit crystallization.

[0080] The resulting mustard yellow crystals and mother liquor weretriturated with isopropanol, which was added in portions to a totalvolume of 1 liter. After an additional 2 hrs. of refrigeration, thesuspension was filtered, washed with isopropanol and diethyl ether toafford IV, 113 gr. (90%), as a mustard yellow powder, mp 182-184° C.,after desiccation in a heated vacuum oven (40-50° C., 0.5 Torr, 14hrs.). The proton NMR spectrum (in perdeuterated acetic acid, 80 MHz)conformed to theory: malate-CH₂, 2.8 ppm, asymmetric doublet, 2H;N,N-(CH₃)₂, 3.1 ppm, singlet, 6H; imido-N—CH₂, 3.7 ppm, degeneratedtriplet, 2H; amino-N—CH₂ and malate-CH, 4.6 ppm, multiplet, 3H; aryl-CH,7.4-8.2 ppm, two apparent multiplets, 5H; OH, 11.4 ppm, singlet, 5H.

[0081] For comparative purposes, between and among organic saltsprepared in this manner and for the purposes of subsequent Example 3,Table 3, crude reaction products were recrystalized from water/ethanol(1.5/4 v/v per gram of salt) using the minimum amount of solvent toeffect dissolution at boil.

[0082] Alternatively, for analytical and bioassay samples,recrystallization was achieved by dissolving the product in water (1/4w/v per gr. of IV) and admixing with a hot mixture of 1/1isopropanol/methanol (1/6 w/v per gr. of IV), heating to boil andremoving any insolubles by hot filtration. Upon cooling first to roomtemperature and then upon chilling for 12 hrs. (0-4° C.), the mass wasfiltered, rinsed with isopropanol followed by diethyl ether and vacuumdesiccated to afford 90 gr. of mustard yellow, rhomboid crystals (85gr., 68% overall yield), mp 184-185° C., homogenous by HPLC, under theconditions shown in Table 1. TABLE 1 HPLC OF MITONAFIDE AND AMONAFIDEMALATE Mobile Flow rate Col Temp Rt Column* Phase** ml/min (° C.) MiTmAMFm Nova Pak C18 4 um 60 A° 17/55/28 1.0 30 >30 NA 3.9 × 150 mm.30/35/35 2.5 40 10.2 13.2 40/25/35 2.5 40 5.1  4.7 Xterra MS C1817/55/28 0.8 40 1.4  3.1 3.5 uM 17/55/28 0.8 30 2.3 NA 3.0 × 150 mm.

Example 2 Synthesis of Amonafide Organic Carboxylic Acid Salts byTitration

[0083] The method of Example 1, illustrating the total synthesis ofamonafide by a sequence of steps in which the organic acid isincorporated at the outset, can be extended to the use of organic acidsother than L-malate. These can be incorporated at any stage in thesynthesis scheme, where appropriate to the reaction flow and consistentwith sound chemical practice. By way of convenient illustration, a panelof salts were readily prepared in semi-automated fashion. It should beunderstood that any analog or congener thereof, with similararalkylamine derived basicity properties, would be equally suitable asan exemplar.

[0084] First a stock solution of amonafide free base, was dispensed intoindividual reaction vials so as to provide a defined amount of basicsubstrate. It was then titrated with a second solution containing onestoichiometric equivalent of an appropriate organic carboxylic acid,whose acidity is consistent with an aqueous pKa value of not less than3. The resulting mixture was warmed in order to effect completedissolution and neutralization of the species reacting ionically, andallowed to deposit the resulting salts as products upon cooling. Thesesolutions may also be concentrated, prior to cooling, in order tooptimize the reaction yield. However, for optimal results the reactionsolvent in this manipulation should be selected so that the reactantsare individually more soluble than their ionic combination.

[0085] In an illustrative example, the free base material wassynthesized according to Brana et al in U.S. Pat. No. 5,183,821. Analiquot was dissolved in boiling anhydrous ethanol at a concentration of1 gram per 20 ml. 10 ml of solution prepared in this manner contains1.765 mMol of material and would, therefore, become neutralized by anequivalent amount of an appropriate organic carboxylic acid in order toafford a monovalent organic salt. Since amonafide is divalent, intheory, it can also be titrated with two equivalents of organic acid toafford a divalent salt. Thus, the number of acid equivalents that can beadded should at least match the calculated minimum number of basicequivalents in the intercalator free drug and not exceed the maximumsuch number.

[0086] For the purpose of this example, however, salt formation has beenrestricted to monoequivalents, and, therefore, solutions of organiccarboxylic acids containing 1.765 mMol in 10-20 ml of water at boil wereprepared and added individually to each of several replicate 100 mlportions of the drug free base at the concentration and volume justdescribed. After bringing the mixed solutions again up to boil, toinsure complete dissolution of all ingredients, they were then left tocool at room temperature and refrigerated overnight, whereupon theresulting crystalline salts were harvested by filtration, dried byrinsing with diethyl ether, and desiccated under vacuum.

[0087] Table 2 provides a listing of the organic carboxylic acids thatwere used to titrate amonafide in order to produce the correspondingcrystalline monovalent salt. Salts obtained in this manner may becharacterized variously by chromatography for chemical homogeneity, andby NMR or mass spectrometry for purposes of structural characterization,as described herein above in Example 1. Characterization by elementalcomposition also affords a convenient method for identity verification.As shown in Table 2, the observed compositions in Part I and thetheoretical elemental compositions in Part II are closely matched,demonstrating that the reaction products constitute an equimolaraddition of each reactant, as would be anticipated for any suchmonovalent adducts of an organic base and an organic acid. TABLE 2AMONAFIDE COMPOSITION OF SALTS ORGANIC SALT Acid C H N O Part I ObservedA) succinic 59.54 5.89 10.13 24.44 B) maleic 60.37 5.10 10.23 24.30 C)fumaric 59.89 5.52 10.20 24.39 D) citric 54.87 5.78 8.30 31.05 E)L-tartaric 56.27 4.95 9.23 29.55 F) L-aspartic 56.80 6.02 13.78 23.40 G)pyruvic 62.45 5.34 11.00 21.21 H 2-oxoglutaric 58.12 5.30 10.40 26.18Part II Calculated A) succinic 59.84 5.78 10.47 23.91 B) maleic 60.145.30 10.52 24.03 C) fumaric 60.14 5.30 10.52 24.03 D) citric 55.58 5.308.84 30.29 E) L-tartaric 55.42 5.35 9.69 29.53 F) L-aspartic 57.69 5.8113.45 23.05 G) pyruvic 61.45 5.70 11.31 21.54 H 2-oxoglutaric 58.74 5.409.79 26.08

[0088] Further confirmation of structural identity and purity was alsoobtained in each instance by NMR analysis in perdeutero acetic acid, thesalt of amonafide and succinic acid being representative, in so far asit is a comparable analog to the malate salt prepared in Example 1.Thus, the salt shown prepared according to Table 2, Part I, entry A, asthe monoequivalent combination of amonafide and succinic acid showed aproton NMR spectrum (in perdeuterated acetic acid, 80 MHz) conforming totheory: succinate-CH₂, 2.7 ppm, singlet, 6H; N,N-(CH₃)₂, 3.1 ppm,singlet, 6H; imido-N—CH₂, 3.7 ppm, degenerated triplet, 2H; amino-N—CH₂,4.6 ppm, degenerated triplet, 2H; aryl-CH, 7.4-8.3 ppm, two apparentmultiplets, 5H; OH, 11.4 ppm, singlet, 4H.

[0089] Although this example illustrates the use of several chiralmolecules, as for example in entries E-F, it follows that suitableresults for purposes of salt formation can be obtained with thecorresponding racemic form or alternate antipodes of such acids. Thus,the selection of the L-enantiomers in this instance should not be takenas a restriction of the teaching; but, rather, as a case in point thatwould be understood by those practiced in the art to be the mostcommonly available such organic carboxylic acid forms suitable forpurposes of biological experimentation.

Example 3 Organic Salts of Amonafide are More Readily Purified than HCLor Methanesulfonic Acid Salts of Amonafide Independent of Method ofSynthesis

[0090] I. Direct Synthesis Amonafide Monohydrochloride Salt by theMethod of Example 1

[0091] 1. Preparation of Mitonafide Monohydrochloride (FW 349.82)

[0092] Reactants:

[0093] (I) 3-nitro-1,8-nitronaphthalic anhydride, (FW 243.18, CAS3027-38-1, purity 99%, ACROS, cat. # 27873-0250);

[0094] (II) N,N-dimethylethylenediamine (FW 88.15, CAS 108-00-9, purity99%, ACROS cat. # 11620-100);

[0095] 6.0 N Hydrochloric acid (FW 36.45 CAS 7647-01-0, purity 99.9%ACROS cat. # 61327-0010

[0096] Synthetic Procedure:

[0097] 10 gr. (0.041 mol, 1 eq.) of the anhydride (I) were combined with130 ml of anhydrous ethanol in a 0.3L 3-neck round bottom flask fittedwith an adding funnel and mechanical paddle stirrer. While vigorouslystirring the suspension, a solution of 4 gr (0.045 mol, 1.1 eq.) of thediamine (II) in 10 ml of anhydrous ethanol was added as a rapid drip.Stirring was continued for 12 hours (overnight), and thereafter themixture was brought to reflux for 1 hour.

[0098] Upon cooling to an internal temperature of 80° C., a pre-warmed(also to 80° C.) solution of 7.5 ml of 6.0 N HCl (0.045 mol, 1.1 eq.)and 2.5 ml of ethanol was added in one portion to the reaction flask andstirring continued for 3 hours. Stirring was stopped and when thereaction reaches room temperature, the crude mitonafidemonohydrochloride was recovered by filtration. The solids wereresuspended in 0.1 liter of anhydrous isopropanol, refiltered, andrinsed with diethyl ether. They were then transferred to a drying dish,triturated mechanically and vacuum desiccated in a drying oven at 0.1Torr with heating to 30° C. for 12 hours.

[0099] A tan solid (11.9 gr., 83% yield) was obtained, which onrecrystallization from water ethanol, 2:1 v/v, afforded 8.3 gr, mp180-183° C. homogenous by HPLC, under the conditions described inExample I.

[0100] 2. Preparation of Amonafide Monohydrochloride (FW 319.79)

[0101] A solution of 10.5 gr. (0.03 mole) mitoriafide monohydrochloridewas suspended in 0.1 liter of deionized, degassed water under an argonblanket in a Parr hydrogenation pressure bottle. 0.15 gr. of 10% Pd/Care added, and the mixture was then evacuated and purged with hydrogengas (three times), then connected to a Parr apparatus and pressurizedwith hydrogen gas to 15 psi. The reaction was vigorously shaken at roomtemperature becoming a yellow solution instead of a tan coloredsuspension within two hours. It was left to hydrogenate for anadditional 12 hours (overnight).

[0102] After evacuative removal of the hydrogen headspace andreplacement with nitrogen, the reaction mixture was stirred with 4 gr.of activated carbon, warmed to ° C., and passed in a Buchner funnelthrough filter paper overlayed with pre-washed Celite filtration aid.The filtrate was concentrated in a 0.25 liter round bottom flask, underreduced pressure in a rotary evaporator with a heating bath thermostatedto 50° C. When a thin crust had begun to form along the meniscus of thesyrupy concentrate, which weighed between 23-24 gr, the flask wasrefrigerated (4° C.) for 12 hrs (overnight) to permit crystallization.

[0103] The resulting mustard yellow crystals and mother liquor weretriturated with isopropanol, which was added in portions to a totalvolume of 0.1 liter. After an additional 2 hours of refrigeration, thesuspension was filtered, washed with isopropanol and diethyl ether toafford 7.9 gr. (81%), as a mustard yellow powder, mp 184-88° C., afterdesiccation in a heated vacuum oven (40-50° C., 0.5 Torr, 14 hrs.).However, the material was non-homogenous by HPLC, under the conditionsshown in Table 1, revealing a main fraction consisting 91% of the peakareas and at last 4 additional impurities ranging in areas from 0.5 to4.5% of the total analyte areas.

[0104] Recrystallization was achieved by dissolving the product in water(1/20 w/v per gr.) and admixing with a hot mixture of 1/1isopropanol/methanol (1/20 w/v), heating to boil and removing anyinsolubles by hot filtration. Upon cooling first to room temperature andthen upon chilling for 12 hrs. (0-4° C.), the mass was filtered, rinsedwith isopropanol followed by diethyl ether and vacuum desiccated toafford mustard yellow needles (6.3 gr., 65% overall yield), mp 289-290°C. Chromatographic analysis revealed the recrystallized material to be97.3% pure and containing an impurity of 1.6%, consisting of theincompletely hydrogenated. A second recrystalization afforded 4.2 (52%overall yield) grams of material with a purity of 99.1%.

[0105] As shown in Table 3, the yield and the need for additionalrecrystallization steps offer a clear contrast to the results of thecongeneric synthesis of organic acid salts of amonafide, e.g. amonafideL-malate from mitonafide L-malate. The latter affords purer product infewer steps, and with a lower solvent volume.

[0106] II. Preparation of Amonafide Malate by the Method of Example 1

[0107] Amonafide malate, amonafide fumarate, amonafide maleate,amonafide malonate and amonafide hemisuccinate were prepared accordingto the method of Example 1. In the case of succinic acid, the organicacid was dispensed as a half-equivalent in order to generate mitonafidehemisuccinate. This method is illustrated schematically in FIG. 1 foramonafide malate. Crude product that was isolated from the hydrogenationreaction mixture was analyzed for purity by high pressure liquidchromatography (HPLC) according to the conditions described in Table 1above. The crude product was recrystallized from water/ethanol (1.5/4v/v) using the minimum amount of solvent required to dissolve the crudeproduct at boil, which is shown in the last column of Table 3 for eachsalt. The recrystallized product was then analyzed for purity by HPLC.The purity for the isolated and recrystallized salts are reported belowin Table 3.

[0108] III. Preparation of Organic Acid Salts of Amonafide by EitherMethod of Example 1 or Method of Example 2

[0109] As shown in the second column of Table 3 below, fumarate, malate,maleate, malonate and hemi-succinate salts of amonafide were obtainedaccording to the procedures described in Example 1 by in situ formationof the corresponding mitonafide salt, followed by catalytichydrogenation. Also, the adipate, aspartate, citrate, glycolate, malate,oxoglutarate, pyruvate, salicylate, succinate, and tartrate salts ofamonafide were obtained according to the procedures described in Example2 from the crude amonafide free base prepared as described above in theprevious paragraph. The purities of the crude salts were assessed byHPLC according to the conditions described in Table 1 above. The crudesalts were then recrystallized from the minimum amount of water/ethanol(1.5/4 v/v) using the minimum amount of solvent required to dissolve thecrude product at boil, which is shown in the last column of Table 3 foreach salt. The resulting purified salts were again assessed for purity.The purity for the crude and recrystallized salts are shown in Table 3below.

[0110] IV. Preparation of the Hydrochloride Salt and MethanesulfonicAcid Salt of Amonafide the Method of Brana et al., U.S. Pat. No.5,420,137

[0111] 1. Preparation of Amonafide Free Base

[0112] Amonafide free base was prepared according to proceduresdisclosed in U.S. Pat. No. 5,183,821 to Brana et al. Exemplaryconditions, at {fraction (1/100)}^(th) the reported scale, are asfollows: 30 g of2-(2-dimethylaminoethyl)-5-nitrobenzo[d,e]isoquinoline-1,3-dione and0.75 gr. of 10% palladium on carbon in 750 milliliters of ethanol wererefluxed with stirring until complete dissolution of the nitro compound.Thereupon 40 milliliters of hydrazine hydrate, meeting currentcommercial specifications of 60% anhydrous hydrazine equivalence, wereadded slowly over a 30 minute period. When addition was complete, refluxand stirring were continued for 3.5 hours, followed by filtration whilestill hot. The reaction mixture was left overnight to reach roomtemperature. The crystalline product was then filtered to affordamonafide free base.

[0113] 2. Preparation of Amonafide Hydrochloride and MethanesulfonicSalts.

[0114] The hydrochloride and methanesulfonic acid salts of amonafidewere prepared according to U.S. Pat. No. 5,420,137. Exemplary proceduresare as follows: 3 g of crude amonafide free base, obtained as describedin the previous two paragraphs, above, were dissolved under reflux in 60ml of virtually anhydrous ethanol. Subsequently 1 ml of 35% (1equivalent, estimated) strength hydrochloric acid was added dropwisewhile shaking vigorously. After cooling, the resulting crystals werefiltered off and washed with 10 ml of anhydrous ethanol. Themethanesulfonic acid salt was obtained using the same procedure, butreplacing hydrochloric acid with 0.8 ml of methanesulfonic acid. Thepurity was assessed by HPLC using the conditions described in Table 1.The isolated products were then recrystallized from water/ethanol (1.5/4v/v) using the minimum amounts of solvent required to dissolve the crudeproduct at boil, which are shown for each salt in Table 3. The resultingpurified salts were again assessed for purity. The results are shown inTable 3 below.

[0115] V. Preparation of the Hydrochloride Salt of Amonafide by theMethod of Zee-Chang et al., U.S. Pat. No. 4,614,820

[0116] The hydrochloride salt of amonafide was obtained by the method ofZee-Chang et al., U.S. Pat. No. 4,614,820. Exemplary conditions are asfollows: a mixture of the hydrochloride salt ofN-(dimethylaminoethyl)-3-nitro-1,8-naphthalimide and 10%palladium-on-charcoal in water was hydrogenated at room temperatureunder 40 lbs/in² of hydrogen for one hour. The theoretical amount ofhydrogen was absorbed. To the mixture was added 3 ml of concentratedhydrochloric acid. It was then filtered and the filtrate was evaporatedto dryness under reduced pressure. The residue was triturated with 30 mlof absolute ethanol and filtered. The resulting solid was then washedwith ether and dried. The purity was assessed by HPLC using theconditions described in Table 1. The crude product was thenrecrystallized from water/ethanol (1.5/4 v/v) using the minimum amountof solvent (40 mL/g) required to dissolve the crude product at boil. Theresulting purified salts were again assessed for purity. The results areshown in Table 3 below.

[0117] VI. Indirect Synthesis of Amonafide Monohydrochoride via SaltExchange from Presursor Amonafide Malate

[0118] 1. Preparation of Amonafide Free Base.

[0119] A batch of amonafide L-malate was prepared in accordance with themethod of Example I. The material so obtained was 99.4%chromatographically pure on a 10 gram synthetic scale, consistent withprevious findings on the larger scale. The material was then convertedto the free base amonafide, as follows: 10 grams of the L-malate saltwere dissolved in 100 ml distilled water and titrated with vigorousstirring to pH 7 by the addition of ¼ concentrated ammonium hydroxide.The large mass of yellow needles that formed was filtered by suction,washed with water, and recrystalized from ethanol to afford 5.8 gr. Of99.8% chromatographically pure amonafide free base, mp 172° C. (MW283.3, 93% yield).

[0120] 2. Preparation of Amonafide Monohydrochloride

[0121] The free base was converted into the monohydrochloride bysuspending 5 gr in 10 ml of water and adding 1 equivalent of 6.0 normal(2.9 ml) with vigorous stirring and warming. Upon dissolution, 20 ml ofboiling ethanol are added slowly to the clouding point, and the mixtureallowed to cool and deposit crystals, which are then harvested byfiltration at room temperature, rinsed with ethanol and ether, andvacuum dried to afford 5.4 grams of product, mp 292° C. The amonafidemonohydrochloride prepared by the method of salt exchange was found tobe 99.2% chromatographically pure and free of any detectableN-hydroxylamine. Notwithstading this level of purity, the amonafidemonohydrochloride prepared by salt exchange showed low solubility whenincluded in the test panel shown in Tables 3 and 4. TABLE 3 Purity (fromPurity reaction (after 1 × Solvent Salt Method pot) crystallization)(ml/g) Amonafide Example 1 92% 99.3% 7.5 Malate Amonafide Example 2 98%99.6% 7.5 Malate Amonafide HCl U.S. Pat. No. 76%   94% 40 5,420,137 toBrana et al. Amonafide HCl U.S. Pat. No. 74%   92% 40 4,614,820 toZee-Cheng, et al. Amonafide HCl Example 1 93% 97.3% 40 Amonafide HClSalt N/A 99.2% 40 exchange Amonafine U.S. Pat. No. 86%   93% 25methanesulfonate 5,420,137 to Brana et al Amonafide Example 2 90% 99.2%20 tartrate Amonafide Example 2 89%   98% 25 Adipate Amonafide Example 278% 98.7% 30 Aspartate Amonafide Example 2 92% 99.1% 25 CitrateAmonafide Example 1 76% 98.9% 25 Fumarate Amonafide Example 2 92% 99.3%12.5 glycolate Amonafide Example 1 70% 99.2% 20 maleate AmonafideExample 1 74%   97% 20 malonate Amonafide 2- Example 2 77% 98.1% 30oxoglutarate Amonafide Example 2 84% 99.5% 20 pyruvate Amonafide Example2 87% 98.7% 35 salicylate Amonafide Example 1 88% 98.7% 15hemi-succinate* Amonafide Example 2 93% 99.2% 20 succinate Amonafide L-Example 2 90% 99.2% 20 tartrate

[0122] As is evident from the data shown in Table 3, the purity ofrecrystallized organic acid salts of amonafide was in every casesignificantly greater than the corresponding recrystallized hydrochloricacid salt and methanesulfonic acid salt. Moreover, in comparing themalate to the HCl moieties, the recrystallization volume forpurification of the malate salt was times smaller, indicating bothgreater solubility in hydroxylic media and also greater ease of handlingin a concentrated solution. The glycolate and hemisuccinate salts werealso significantly more soluble than either the HCl or methanesulfonicacid salt.

EXAMPLE 4 Organic Acid Salts of Amonafide are More Soluble in Water thanthe Corresponding Hydrochloride Salt Independent of Method of Synthesis

[0123] A number of organic acid amonafide salts of the present inventionwere tested for their solubility in aqueous medium and compared with thesolubility of the corresponding hydrochloride and methansolufonic acidsalts. Specifically, solubility assessment (in duplicate) for the saltsurvey was conducted by dissolving, or attempting to dissolve, 50, 100and 200 mg of each salt in 1 ml of USP water or 1 ml of isotonic salineby vortexing at ambient temperature. Solutions were allowed toequilibrate for 1 hour, their dissolution status noted, and thenrefrigerated for an additional hour (at 2° C.). Thereafter, the sampleswere returned to room temperature and examined for precipitate formationand allowed to equilibrate for another hour until a final inspection fordissolution status. Results are presented in Table 4. TABLE 4 Amount ofAMONAFIDE Amonafide Dissolution SALT Salt (mg) Solvent Timepoint StatusL-Aspartate 100 0.9% Sodium 0 hours Soluble chloride (saline) DistilledWater 1 hours Soluble 0 hours Soluble 1 hours Soluble 200 0.9% Sodium 0hours Partially soluble chloride (saline) Distilled Water 1 hoursPartially soluble 0 hours Partially soluble 1 hours Partially solubleCitrate 50 0.9% Sodium 0 hours Insoluble chloride (saline) DistilledWater 1 hours Insoluble 0 hours Insoluble 1 hours Insoluble Fumarate 500.9% Sodium 0 hours Insoluble chloride (saline) Distilled Water 1 hoursInsoluble 0 hours Insoluble 1 hours Insoluble Maleate 50 0.9% Sodium 0hours Insoluble chloride (saline) Distilled Water 1 hours Insoluble 0hours Insoluble 1 hours Insoluble Malonate 50 0.9% Sodium 0 hoursSoluble chloride (saline) Distilled Water 1 hours Soluble 0 hoursSoluble 1 hours Soluble 100 0.9% Sodium 0 hours Insoluble chloride(saline) Distilled Water 1 hours Insoluble 0 hours Insoluble 1 hoursInsoluble Methanesulfonate 200 0.9% Sodium 0 hours Soluble chloride(saline) Distilled Water 1 hours Soluble 0 hours Soluble 1 hours Soluble2-Oxo-glutarate 50 0.9% Sodium 0 hours Soluble chloride (saline)Distilled Water 1 hours Soluble 0 hours Soluble 1 hours Soluble 100 0.9%Sodium 0 hours Insoluble chloride (saline) Distilled Water 1 hoursInsoluble 0 hours Insoluble 1 hours Insoluble Succinate 50 0.9% Sodium 0hours Insoluble chloride (saline) Distilled Water 1 hours Insoluble 0hours Soluble 1 hours Soluble 100 0.9% Sodium 0 hours Insoluble chloride(saline) Distilled Water 1 hours Insoluble 0 hours Insoluble 1 hoursInsoluble mono- 50 0.9% Sodium 0 hours Partially soluble Hydrochlorideby chloride (saline) the method of Distilled Water 1 hours Partiallysoluble Example 1 0 hours Partially soluble 1 hours Partially soluble100 0.9% Sodium 0 hours Insoluble chloride (saline) Distilled Water 1hours Insoluble 0 hours Partially soluble 1 hours Insoluble 200 0.9%Sodium 0 hours Insoluble chloride (saline) Distilled Water 1 hoursInsoluble 0 hours Insoluble 1 hours Insoluble mono- 50 0.9% Sodium 0hours Partially soluble Hydrochloride by chloride (saline) the method ofsalt Distilled Water 1 hours Partially soluble exchange 0 hoursPartially soluble 1 hours Partially soluble 100 0.9% Sodium 0 hoursInsoluble chloride (saline) Distilled Water 1 hours Insoluble 0 hoursPartially soluble 1 hours Insoluble 200 0.9% Sodium 0 hours Insolublechloride (saline) Distilled Water 1 hours Insoluble 0 hours Insoluble 1hours Insoluble mono- 50 0.9% Sodium 0 hours Partially solubleHydrochloride chloride (saline) by the method Distilled Water 1 hoursPartially soluble of Brana et al. 0 hours Partially soluble 1 hoursPartially soluble 100 0.9% Sodium 0 hours Insoluble chloride (saline)Distilled Water 1 hours Insoluble 0 hours Partially solubledi-Hydrochloride 50 0.9% Sodium 0 hours Insoluble chloride (saline)Distilled Water 1 hours Insoluble 0 hours Partially soluble 1 hoursPartially soluble 100 0.9% Sodium 0 hours Insoluble chloride (saline)Distilled Water 1 hours Insoluble 0 hours Insoluble 1 hours InsolubleL-Malate 50 0.9% Sodium 0 hours Soluble chloride (saline) DistilledWater 1 hours Soluble 0 hours Soluble 1 hours Soluble 100 0.9% Sodium 0hours Soluble chloride (saline) Distilled Water 1 hours Soluble 0 hoursSoluble 1 hours Soluble 200 0.9% Sodium 0 hours Soluble chloride(saline) Distilled Water 1 hours Soluble 0 hours Soluble 1 hours SolublePyruvate 50 0.9% Sodium 0 hours Soluble chloride (saline) DistilledWater 1 hours Soluble 0 hours Soluble 1 hours Soluble 100 0.9% Sodium 0hours Soluble chloride (saline) Distilled Water 1 hours Soluble 0 hoursSoluble 1 hours Soluble 200 0.9% Sodium 0 hours Partially Solublechloride (saline) Distilled Water 1 hours Partially Soluble 0 hoursSoluble 1 hours Soluble Tartrate 50 0.9% Sodium 0 hours Soluble chloride(saline) Distilled Water 1 hours Soluble 0 hours Soluble 1 hours Soluble100 0.9% Sodium 0 hours Soluble chloride (saline) Distilled Water 1hours Soluble 0 hours Soluble 1 hours Soluble 200 0.9% Sodium 0 hoursSoluble chloride (saline) Distilled Water 1 hours Soluble 0 hoursSoluble 1 hours Soluble Glycolate 50 0.9% Sodium 0 hours Solublechloride (saline) Distilled Water 1 hours Soluble 0 hours Soluble 1hours Soluble 100 0.9% Sodium 0 hours Soluble chloride (saline)Distilled Water 1 hours Soluble 0 hours Soluble 1 hours Soluble 200 0.9%Sodium 0 hours Soluble chloride (saline) Distilled Water 1 hours Soluble0 hours Soluble 1 hours Soluble

[0124] From the data in Table 4, it is evident that a number of organicacid salts of amonafide are more soluble in distilled water and insaline at high concentration than either of the correspondinghydrochloride salt forms. These latter include the aspartate, malate,pyruvate, oxo-glutarate and tartrate. It is also notable that regardlessof method of preparation, the monohydrochloride salts show consistentlyinferior solubility when compared to representative organic salts.

Example 5 Amonafide Malate has Anti-Cancer Activity In Vitro

[0125] Amonafide Malate was tested for cytotoxic activity in a panel ofthree cell lines, MCF7 (Breast), H460 (Non-Small Cell Lung Cancer) andSF268 (Glioma). These cell lines are used by the National CancerInstitute: Developmental Therapeutics Branch in their preliminaryscreening process for antineoplastic agents. Subsequently, AmonafideMalate was tested against a larger panel of breast, lung and colon celllines Breast: MCF-7, BT474, MDA-231, T47D and SKBr3 Colon: HT29, HCT116and COLO205 Lung: H460, H23 and A549 Prostate: DU-145, PC-3 and LNCaP

[0126] The following protocol was used:

[0127] Cells were grown in RPMI 1640 medium containing 5% fetal bovineserum and 2 mM L-glutamine. Dependent upon cell doubling time, between5,000 and 40,000 cells were inoculated into 96 well microtiter plates ina volume of 100 uL per well. The plates were incubated at 37° C., 5%CO₂, 95% air, and 100% relative humidity for 24 hours prior to theaddition of the experimental drug. After the, 24 hour incubation, two(2) plates of each cell line were fixed in situ with trichloroaceticacid (TCA) to establish the cell population at time of drug addition(Tz).

[0128] Prior to use, the experimental drugs were solubilized in dimethylsulfoxide at 400-fold the desired final maximum test concentration andfrozen. At time of drug addition, an aliquot of frozen concentrate wasthawed and diluted to twice the desired final maximum test concentrationwith complete medium containing 50 ug/ml gentamicin. An additional four(4) 10-fold or 12 log serial dilutions was made for a total of five (5)drug concentrations plus a control. An aliquot of 100 ul of each drugdilution was added to the appropriate well that already contains 100 ulof medium containing the cells. The plates were then incubated for 48hours at 37° C., 5% CO₂; 95% air and 100% relative humidity.

[0129] The number of viable cells was estimated by either a SRB or MTTcalorimetric assays.

[0130] For SRB assay, cells were fixed in situ by the gentle addition of50 ul cold (w/v) TCA (final concentration, 10% TCA) and incubated for 60minutes at 4° C. The supernatant was discarded, plates washed five (5)times with tap water and air dried. Then Sulforhodamine B (SRB) solution(100 μl) at a 0.4% (w.v.) in 1% acetic acid was added to each well, andthe plates incubated for 10 minutes at room temperature. Unbound dye wasremoved by washing five (5) times with 1% acetic acid and the plateswere air dried. The bound stain was solubilized with 10 mM trizmabaseanad the absorbance was read on an automated plate reader at awavelength of 515 nm.

[0131] The MTT assay measures the ability of viable cells to reduce atetrazolium salt (MTT) to an insoluble form, a formazan salt. For MTTanalysis 20 μl of MTT solution (5 mg/ml in PBS) was added to each wellcontaining cells. The plate was incubated at 37° C. for 5 hours. Mediawas removed with needle and syringe. 200 μl of DMSO was added to eachwell and pipetted up and down to dissolve crystals. Plate was placedinto a 37° C. incubator for 5 minutes to dissolve air bubbles.Absorbance was read on an automated plate reader at a wavelength of 550nm.

[0132] Percent net growth inhibition, as shown in FIGS. 2-7, wascalculated using the seven absorbance measurements: absorbance by cellsat time zero (Tz), absorbance by cells grown in the absence of drug (C),and absorbance of cells grown at the five drug concentrations (Ti). Thefollowing expression was used:

% Net Growth=[(Ti−Tz)/(C−Tz)]×100 if Ti was greater than or equal to Tz.

% Net Growth=[(Ti−Tz)/Tz]×100 if Ti was less than Tz.

[0133] Using the plot of Percent Net Growth as a function of drugconcentration, lethality concentration “50%” (LC₅₀), total growthinhibition (TGI) and growth inhibition “50%” (GI₅₀) were determined asfollows.

[0134] TGI was determined as the concentration of Amonafide malate atwhich Percent Net Growth was 0%. GI₅₀ was determined as theconcentration of Amonafide malate at which Percent Net Growth was 50%.LC₅₀ was determined as the concentration of Amonafide malate at whichPercent Net Growth was −50%.

[0135] As can be seen in FIG. 2, Amonafide malate significantly affectedthe growth rate of cell lines H-460 (non-small cell lung carcinoma),SF-268 (glioblastoma) and MCF-7 (breast cancer) resulting in GI₅₀ ofbetween 4 and 8 μM. The data presented in fable 5 summarizes the resultsshown in FIG. 2 TABLE 5 H460 SF268 MCF7 GI₅₀ (M) 4.11E−06 7.70E−065.16E−06 TGI (M) 0.96E−04 1.54E−04 1.10E−04 LC₅₀ (M) 1.70E−04 8.42E−046.62E−04

[0136] FIGS. 3 to 7 present data for panels of cell lines derived frombreast cancer (FIG. 3), colorectal cancer (FIG. 4), lung cancer (FIG. 5,using SRB assay, and FIG. 6, using MTT assay) and prostate cancer (FIG.7). As can be seen, reduction of net growth by as much as 50% or betterwas achieved at Amonafide malate concentrations between about 5 andabout 10 μM.

Example 6 Amanofide has Anti-Cancer Activity In Vivo

[0137] Amonafide Malate was tested for in vivo anti-cancer activity inmodels of three different solid tumor types, MCF7 and MDA-231 (Breast),COLO205 (Colorectal) and PC3 (Prostate). The in vivo activity was testedusing the Hollow Fiber methodology developed by the National CancerInstitute: Developmental Therapeutics Branch for use in theirantineoplastic agent screening program. Specifically, the anti-canceractivity of amonafide L-malate was tested by the following protocol.

[0138] Polyvinylidene fluoride (PVDF) hollow fibers were used. Thefibers were individually flushed and filled with 70% ethanol andincubated in 70% ethanol at room temperature for a minimum of 96 hour.Following three washes with deionized water, the fibers were filled withand placed into a pan of deionized water for sterilization byautoclaving. Then the fibers were stored in water at 4° C. until used.

[0139] Mice were grouped into 4 groups, two control groups with 6 miceper group and 2 experimental groups with 3 mice per group. Theexperimental groups were as follows:

[0140] Group A: Control, hollow fibers without cells

[0141] Group B: Control, hollow fibers for 3 cell lines without drugtreatment

[0142] Group C: hollow fibers for 3 cell lines+Amonafide Malate

[0143] Anesthesia was induced in mice by ketamine/acepromazine/xylazineinjected intraperitoneally (i.p.) For the intraperitoneal (i.p.)implants, a small incision was made through the skin and musculature ofthe dorsal abdominal wall, the fiber samples were inserted into theperitoneal cavity in a craniocaudal direction and the incision wasclosed with a skin staple. For subcutaneous (s.c.) implants, a smallskin incision was made at the nape of the neck to allow insertion of an11 gauge tumor implant trocar. The trocar, containing the hollow fibersamples, was inserted caudally through the subcutaneous tissues and thefibers were deposited during withdrawal of the trocar. The skin incisionwas closed with a skin staple. Each mouse was host of 6 samples,representing 3 tumor cells lines each of which was cultured in the 2physiologic compartments (i.p. and s.c.).

[0144] In a first series of experiments, Amonafide Malate (29.4 mg/kg)was injected intraperitoneally (i.p.), once daily on days 3-8. In asecond series of experiments, Amonafide Malate was administered i.p.twice-daily at either 15 mg/kg or 29 mg/kg.

[0145] On the day following the last drug injection, the animals weresacrificed and the hollow fibers extracted. The hollow fibers were thensubjected to the stable endpoint MTT assay as described in Example 5.The optical density of each sample is determined spectrophotometricallyat 540 nm and the mean of each treatment group is calculated. Thepercent net growth for each cell line in each treatment group iscalculated as described in Example 5 and compared to the percent netgrowth in the vehicle treated controls (Group B) using the formula belowto determine the percent growth inhibition:

% growth inhibition=(Net Growth_(Control)−Net Growth_(Treated))/NetGrowth_(Control))*100

[0146] Individual mouse body weights were recorded daily and animalswere monitored daily for general health for 6 days. It was not necessaryto sacrifice any animals in a CO₂ chamber prematurely as a result of abody weight loss of greater than 20%, or as a result of evidence ofother signs of toxicity.

[0147]FIG. 8 and Table 6 show data for once-daily administration of 29.4mg/kg of Amonafide Malate. FIGS. 9 and 10 and Tables 7 and 8 show datafor twice-daily administration of Aminofide Malate at either 15 mg/kg or29 mg/kg.

[0148] As presented in Tables 6-8 and FIG. 10, Amonafide malate showedconsiderable activity, resulting in percent growth inhibition of above100% (indicating lethality for cancerous cells) for cell lines MCF-7 andMDA-231 (breast cancer). Significant percent growth inhibition wasachieved in cell lines PC-3 (prostate) and COLO205 (colorectal cancer).TABLE 6 IP SC % Growth % Growth Inhibition STDEV Inhibition STDEV MCF-7150.2 19.2 116.1 12.1 COLO205 84.6 11.7 80.4 11.7 PC3 71.6 7.3 52.9 10.9

[0149] TABLE 7 I.P. fibers % Growth Inhibition Cell line AmonafideMalate, Amonafide Malate, Type Name 15 mg/kg 29 mg/kg Breast MCF-7 97%124% Breast MDA-231 93% 108% Lung H23 36%  47% Colon COLO205 82%  98%

[0150] TABLE 8 S.C. fibers % Growth Inhibition Cell line AmonafideMalate, Amonafide Malate, Type Name 15 mg/kg 29 mg/kg Breast MCF-7 83%107% Breast MDA-231 79% 113% Lung H23 13%  27% Colon COLO205 50%  76%

[0151] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A compound represented by the followingstructural formula:

wherein: R1 is —(CH₂)_(n)N⁺HR3R4 X⁻ or R1 is —(CH₂)_(n)N⁺HR3R4 X⁻ or—(CH₂)_(n)NR3R4 when R2 is —N⁺HR6R7; R2 is —OR5, halogen, —NR6R7,—N⁺HR6R7 X^(−,) sulphonic acid, nitro, —NR5COOR5, —NR5COR5 or —OCOR5; R3and R4 are independently H, C1-C4 alkyl group or, taken together withthe nitrogen atom to which they are bonded, a non-aromaticnitrogen-containing heterocyclic group; each R5 is independently —H or aC1-C4 alkyl group; R6 and R7 are independently H, C1-C4 alkyl group or,taken together with the nitrogen atom to which they are bonded, anon-aromatic nitrogen-containing heterocyclic group; n is an integerfrom 0-3; and X⁻ is the carboxylate anion of an organic carboxylic acidcompound.
 2. The compound of claim 1 wherein: n is 2; R2 is —NO₂, —NH₂or —NH₃+X⁻; and R3 and R4 are the same and are —H, —CH₃ or —CH₂CH₃. 3.The compound of claim 2 wherein R3 and R4 are —CH₃.
 4. The compound ofclaim 1 wherein X⁻ is the carboxylate anion of a C1-C4 aliphaticmonocarboxylic acid, hydroxy C2-C6 aliphatic monocarboxylic acid, ketoC2-C6 aliphatic monocarboxylic acid, amino C2-C6 aliphaticmonocarboxylic acid, C2-C8 aliphatic dicarboxylic acid, hydroxy C3-C8aliphatic dicarboxylic acid, keto C3-C8 aliphatic dicarboxylic acid,amino C3-C8 aliphatic dicarboxylic acid, C3-C8 aliphatic tricarboxylicacid, hydroxy C4-C10 tricarboxylic acid, keto C4-C10 tricarboxylic acid,amino C4-C10 tricarboxylic acid, an aryl carboxylic acid, C1-C5heteroalkyl monocarboxylic acid or C3-C8 heteroalkyl dicarboxylic acid.5. The compound of claim 1 wherein X⁻ is the carboxylate anion of ahydroxy C2-C6 aliphatic monocarboxylic acid, a keto C2-C6 aliphaticmonocarboxylic acid, a C2-C8 aliphatic dicarboxylic acid, a hydroxyC3-C8 aliphatic dicarboxylic acid, a keto C3-C8 aliphatic dicarboxylicacid, a C3-C8 tricarboxylic acid, a hydroxy C4-C8 tricarboxylic acid, ora keto C4-C8 tricarboxylic acid and the compound is amonafide.
 6. Thecompound of claim 3 wherein X⁻ is the carboxylate anion of a C1-C4aliphatic monocarboxylic acid, hydroxy C2-C6 aliphatic monocarboxylicacid, keto C2-C6 aliphatic monocarboxylic acid, amino C2-C6 aliphaticmonocarboxylic acid, C2-C8 aliphatic dicarboxylic acid, hydroxy C3-C8aliphatic dicarboxylic acid, keto C3-C8 aliphatic dicarboxylic acid,amino C3-C8 aliphatic dicarboxylic acid, C3-C8 aliphatic tricarboxylicacid, hydroxy C4-C10 tricarboxylic acid, keto C4-C10 tricarboxylic acid,amino C4-C10 tricarboxylic acid, an aryl carboxylic acid, C1-C5heteroalkyl monocarboxylic acid or C3-C8 heteroalkyl dicarboxylic acid.7. The compound of claim 1 wherein X⁻ is the carboxylate anion of formicacid, acetic acid, propionic acid, 2-pentenoic acid, 3-pentenoic acid,3-methyl-2-butenoic acid, 4-methyl-3-pentenoic acid lactic acid,glycolic, mandelic acid, oxaloacetic acid, alpha-ketoglutaric acid,pyruvic acid, aspartic acid, glutamic acid, malonic acid, succinic acid,adipic acid, maleic acid, fumaric acid, malic acid, tartaric acid,citric acid or gluconic acid.
 8. The compound of claim 3 wherein X⁻ isthe carboxylate anion of formic acid, acetic acid, propionic acid,2-pentenoic acid, 3-pentenoic acid, 3-methyl-2-butenoic acid,4-methyl-3-pentenoic acid, lactic acid, glycolic, mandelic acid,oxaloacetic acid, alpha-ketoglutaric acid, pyruvic acid, aspartic acid,glutamic acid, malonic acid, succinic acid, adipic acid, maleic acid,fumaric acid, malic acid, tartaric acid, citric acid or gluconic acid.9. The compound of claim 1 wherein the compound is amonafide tartrate,amonafide adipate, amonafide aspartate, amonafide citrate, amonafidefumarate, amonafide glycolate, amonafide maleate, amonafide malonate,amonafide 2-oxoglutarate, amonafide pyruvate, amonafide salicylate,amonafide hemi-succinate or amonafide succinate.
 10. The compound ofclaim 1 wherein X is malate or glycolate.
 11. The compound of claim 1wherein the compound is monovalent.
 12. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent and acompound represented by the following structural formula:

wherein: R1 is —(CH₂)_(n)N⁺HR3R4 X⁻ or R1 is —(CH₂)_(n)N⁺HR3R4 X— or—(CH₂)_(n)NR3R4 when R2 is —N⁺HR6R7; R2 is —OR5, halogen, —NR6R7,—N⁺HR6R7 X^(−,) sulphonic acid, nitro, —NRSCOOR5, —NR5COR5 or —OCOR5; R3and R4 are independently H, C1-C4 alkyl group or, taken together withthe nitrogen atom to which they are bonded, a non-aromaticnitrogen-containing heterocyclic group; each R5 is independently —H or aC1-C4 alkyl group; R6 and R7 are independently H, C1-C4 alkyl group or,taken together with the nitrogen atom to which they are bonded, anon-aromatic nitrogen-containing heterocyclic group; n is an integerfrom 0-3; and X⁻ is the carboxylate anion of an organic carboxylic acidcompound.
 13. The pharmaceutical composition of claim 12 wherein: n is2; R2 is —NO₂, —NH₂ or —NH₃+X⁻; and R3 and R4 are the same and are —H,—CH₃ or —CH₂CH₃.
 14. The pharmaceutical composition of claim 13 whereinR3 and R4 are —CH₃.
 15. The pharmaceutical composition of claim 12wherein X⁻ is the carboxylate anion of a C1-C4 aliphatic monocarboxylicacid, hydroxy C2-C6 aliphatic monocarboxylic acid, keto C2-C6 aliphaticmonocarboxylic acid, amino C2-C6 aliphatic monocarboxylic acid, C2-C8aliphatic dicarboxylic acid, hydroxy C3-C8 aliphatic dicarboxylic acid,keto C3-C8 aliphatic dicarboxylic acid, amino C3-C8 aliphaticdicarboxylic acid, C3-C8 aliphatic tricarboxylic acid, hydroxy C4-C10tricarboxylic acid, keto C4-C10 tricarboxylic acid, amino C4-C10tricarboxylic acid, an aryl carboxylic acid, C1-C5 heteroalkylmonocarboxylic acid or C3-C8 heteroalkyl dicarboxylic acid.
 16. Thepharmaceutical composition of claim 14 wherein X⁻ is the carboxylateanion of a C1-C4 aliphatic monocarboxylic acid, hydroxy C2-C6 aliphaticmonocarboxylic acid, keto C2-C6 aliphatic monocarboxylic acid, aminoC2-C6 aliphatic monocarboxylic acid, C2-C8 aliphatic dicarboxylic acid,hydroxy C3-C8 aliphatic dicarboxylic acid, keto C3-C8 aliphaticdicarboxylic acid, amino C3-C8 aliphatic dicarboxylic acid, C3-C8aliphatic tricarboxylic acid, hydroxy C4-C 10 tricarboxylic acid, ketoC4-C 10 tricarboxylic acid, amino C4-C 10 tricarboxylic acid, an arylcarboxylic acid, C1-C5 heteroalkyl monocarboxylic acid or C3-C8heteroalkyl dicarboxylic acid.
 17. The pharmaceutical composition ofclaim 12 wherein X⁻ is the carboxylate anion of formic acid, aceticacid, propionic acid, 2-pentenoic acid, 3-pentenoic acid,3-methyl-2-butenoic acid, 4-methyl-3-penterioic acid lactic acid,glycolic, mandelic acid, oxaloacetic acid, alpha-ketoglutaric acid,pyruvic acid, aspartic acid, glutamic acid, malonic acid, succinic acid,adipic acid, maleic acid, fumaric acid, malic acid, tartaric acid,citric acid or gluconic acid.
 18. The pharmaceutical composition ofclaim 14 wherein X⁻ is the carboxylate anion of formic acid, aceticacid, propionic acid, 2-pentenoic acid, 3-pentenoic acid,3-methyl-2-butenoic acid, 4-methyl-3-pentenoic acid, lactic acid,glycolic, mandelic acid, oxaloacetic acid, alpha-ketoglutaric acid,pyruvic acid, aspartic acid, glutamic acid, malonic acid, succinic acid,adipic acid, maleic acid, fumaric acid, malic acid, tartaric acid,citric acid or gluconic acid.
 19. The pharmaceutical composition ofclaim 12 wherein X⁻ is malate or glycolate.
 20. A method of treating asubject with cancer comprising the step of administering to the subjectan effective amount of a compound represented by the followingstructural formula:

wherein: R1 is —(CH₂)_(n)N⁺HR3R4 X⁻ or R1 is —(CH₂)_(n)N⁺HR3R4 X or—(CH₂)_(n)NR3R4 when R2 is —N⁺HR6R7; R2 is —OR5, halogen, —NR6R7,—N⁺HR6R7 X⁻ sulphonic acid, nitro, —NR5COOR5, —NR5COR5 or —OCOR5; R3 andR4 are independently H, C1-C4 alkyl group or, taken together with thenitrogen atom to which they are bonded, a non-aromaticnitrogen-containing heterocyclic group; each R5 is independently —H or aC1-C4 alkyl group; R6 and R7 are independently H, C1-C4 alkyl group or,taken together with the nitrogen atom to which they are bonded, anon-aromatic nitrogen-containing heterocyclic group; n is an integerfrom 0-3; and X⁻ is the carboxylate anion of an organic carboxylic acidcompound.
 21. The method of claim 20 wherein: n is 2; R2 is —NO₂, —NH₂or —NH₃+X⁻; and R3 and R4 are the same and are —H, —CH₃ or —CH₂CH₃. 22.The method of claim 21 wherein R3 and R4 are —CH₃.
 23. The method ofclaim 20 wherein X⁻ is the carboxylate anion of a C1-C4 aliphaticmonocarboxylic acid, hydroxy C2-C6 aliphatic monocarboxylic acid, ketoC2-C6 aliphatic monocarboxylic acid, amino C2-C6 aliphaticmonocarboxylic acid, C2-C8 aliphatic dicarboxylic acid, hydroxy C3-C8aliphatic dicarboxylic acid, keto C3-C8 aliphatic dicarboxylic acid,amino C3-C8 aliphatic dicarboxylic acid, C3-C8 aliphatic tricarboxylicacid, hydroxy C4-C10 tricarboxylic acid, keto C4-C10 tricarboxylic acid,amino C4-C10 tricarboxylic acid, an aryl carboxylic acid, C1-C5heteroalkyl monocarboxylic acid or C3-C8 heteroalkyl dicarboxylic acid.24. The method of claim 22 wherein X⁻ is the carboxylate anion of aC1-C4 aliphatic monocarboxylic acid, hydroxy C2-C6 aliphaticmonocarboxylic acid, keto C2-C6 aliphatic monocarboxylic acid, aminoC2-C6 aliphatic monocarboxylic acid, C2-C8 aliphatic dicarboxylic acid,hydroxy C3-C8 aliphatic dicarboxylic acid, keto C3-C8 aliphaticdicarboxylic acid, amino C3-C8 aliphatic dicarboxylic acid, C3-C8aliphatic tricarboxylic acid, hydroxy C4-C10 tricarboxylic acid, ketoC4-C10 tricarboxylic acid, amino C4-C10 tricarboxylic acid, an arylcarboxylic acid, C1-C5 heteroalkyl monocarboxylic acid or C3-C8heteroalkyl dicarboxylic acid.
 25. The method of claim 20 wherein X⁻ isthe carboxylate anion of formic acid, acetic acid, propionic acid,2-pentenoic acid, 3-pentenoic acid, 3-methyl-2-butenoic acid,4-methyl-3-pentenoic acid lactic acid, glycolic, mandelic acid,oxaloacetic acid, alpha-ketoglutaric acid, pyruvic acid, aspartic acid,glutamic acid, malonic acid, succinic acid, adipic acid, maleic acid,fumaric acid, malic acid, tartaric acid, citric acid or gluconic acid.26. The method of claim 22 wherein X⁻ is the carboxylate anion of formicacid, acetic acid, propionic acid, 2-pentenoic acid, 3-pentenoic acid,3-methyl-2-butenoic acid, 4-methyl-3-pentenoic acid, lactic acid,glycolic, mandelic acid, oxaloacetic acid, alpha-ketoglutaric acid,pyruvic acid, aspartic acid, glutamic acid, malonic acid, succinic acid,adipic acid, maleic acid, fumaric acid, malic acid, tartaric acid,citric acid or gluconic acid.
 27. The method of claim 20 wherein X⁻ ismalate or glycolate.
 28. The method of claim 20 wherein cancer isselected from the group consisting of breast cancer, colon cancer, lungcancer, prostate cancer, renal cancer, glioma and leukemia.
 29. Themethod of claim 20 wherein cancer is selected from the group consistingof breast cancer, colon cancer, lung cancer, renal cancer and prostatecancer.